The psychoactive and therapeutic effects of 9-tetrahydrocannabinold (THC), the main psychoactive constituent in marijuana, are mediated by CB1 receptors (CB1R) in the CNS. However, therapeutic use is limited by THC abuse liability and the development of tolerance and dependence with chronic use. At the molecular level, tolerance is mediated by both uncoupling of CB1Rs from G-proteins (desensitization) and/or reduction in the density of CB1Rs (downregulation). Regional differences exist in the development of desensitization/downregulation; however, little is known about the contributors to these differences. In particular, brain regions like the nucleus accumbens (NAC) and caudate-putamen (CPU) that contribute to the reward and motivational properties of THC have reduced sensitivity to desensitization/downregulation and extended time course of the development/recovery of adaptation. These areas are comprised of two populations of medium spiny neurons (MSNs) that have both distinct projections and express either dopamine receptor type 1 (D1) receptors/dynorphin and dopamine type 2 receptors/enkephalin. These areas also have high basal levels of the cAMP-regulated phosphoprotein of Mr = 32,000 (DARPP-32) and high induction of the stable transcription factor FosB following chronic treatment with THC. Desensitization/downregulation in these areas is also dependent on activation of the extracellular signal-regulated kinase (ERK). Protein kinase A (PKA) phosphorylation of DARPP-32 at threonine 34 (T34-DARPP-32) is important for the induction of FosB and the activation of ERK whereas cyclin dependent kinase 5 (CDK5), a target of FosB mediated regulation of transcription, is responsible for the phosphorylation of DARPP-32 at threonine 75 (T75-DARPP- 32). We propose that these proteins are important for the resistance of desensitization/downregulation in the NAC and CPU. The first aim will test the hypothesis that chronic THC increases expression levels of CDK5 and phosphorylation of T75-DARPP-32 while decreasing the phosphorylation of T34-DARPP-32 in the NAC and CPU using western blots. Further, studies to co-localize prodynorphin or [Met] enkephalin with FosB and T75- DARPP-32 will test whether these expression patterns predominate in one MSN population. The second aim will test the effects of FosB on desensitization/downregulation in the NAC and CPU using [35S] GTP3S and [3H] receptor binding, respectively, with either mice transgenically overexpressing FosB predominantly in D1/dynorphin MSNs or mice expressing a dominant negative of FosB (c-Jun) in both populations of MSNs. We predict that chronic THC will increase levels of FosB, CDK5 and T75-DARPP-32, but decrease T34- DARPP-32, predominantly in D1/dynorphin MSNs and that overexpression of FosB will reduce desensitization/downregulation whereas expression of c-Jun will increase adaptations. An understanding of the proteins involved in desensitization/downregulation of CB1Rs will provide new therapeutic targets for the modulation of tolerance to the effects of THC to enhance its therapeutic benefits and reduce abuse liability. PUBLIC HEALTH RELEVANCE: Marijuana and cannabinoid drugs modulate the function of brain regions that control motivation and motor activity. The proposed studies will determine the effects of repeated cannabinoid administration on biochemical messengers that regulate activity in these brain regions.